RNA isolation - purity (Mar/15/2005 )
I have isolated total RNA from human cells using QIagen kit. Potential DNA was digested with DNase delivered by Qiagen in the same kit. And... I have beautiful bands with RNA ONLY and primers for DNA. I excluded any contamination in chemicals, pipets and so on.
What is that?
Too much cell?
DNase which does not work?
Gratefull for any help
P.S. I did this preparation twice - the same result
may you explain little more this point?
how long was your rna extraction? did you always worked in ice?
If I understand your problem, you're able to amplify a DNA sequence (intronic, promoter, something like that?) from your RT. Is that it? In that case, if you discard contamination by amplicons (how was your blank?), I guess that the DNAse didn't work. You don't need much DNA contamination to get amplification by PCR especially if you've got good primers, and you're bound to get some DNA contamination with almost all methods of RNA isolation you may use. Depending on your own ability or experience, the quality and amount of starting material, and the method you use for isolation, you will get lots of contaminating DNA which can be seen when running the RNA samples on a gel, or just really small amounts that may not even be detected by PCR. In your case, I would check another DNase, or incubate longer with the same one. Cheers!
I am also very curious about a new DNase (TURBO DNA free kits,cat#1907 )from Ambion. Can anyone tell me it works really better than its precursor DNase I? thanks in advance
I have used the Turbo kit, and it uses a nice inactivation system which consists of a sludge, I have used conventional DNAse but the Turbo kit works fine in my hands and I am very happy with it.
Thanks a lot, Nick.